Rational design and implementation of a chemically inducible heterotrimerization system

Abstract

Chemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools such as the FK506-binding protein–FKBP–rapamycin-binding– (FKBP–FRB)–rapamycin system have been widely used to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) does not exist, due to inherent challenges in designing a chemical that simultaneously binds three proteins with high affinity and specificity. Here, we developed CIT by rationally splitting FRB and FKBP. Cellular and structural datasets showed efficient trimerization of split pairs of FRB or FKBP with full-length FKBP or FRB, respectively, by rapamycin. CIT rapidly induced tri-organellar junctions and perturbed intended membrane lipids exclusively at select membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells to address cell biology questions otherwise intractable and engineer cell functions for future synthetic biology applications.

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Fig. 1: Rational CIT design and sFRB overexpression in cells.
Fig. 2: Characterization of FKBP recruitment extent and kinetics by sFRB after rapamycin addition.
Fig. 3: Crystal structure of the split and unsplit FKBP–rapamycin–FRB (T2098L) complexes.
Fig. 4: CIT-based recruitment of cytosolic protein to ER–PM and ER–mitochondria MCS.
Fig. 5: Tri-organellar junction formation by CIT.
Fig. 6: CIT induces local PI(4,5)P2 depletion at ER–PM MCS.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank L. Bertozzi and S. Thompson for help with plasmid generation. We thank R. DeRose, X.Y. Zhou and Y. Nihongaki for proofreading the manuscript. We thank H. Niwa (RIKEN), N. Sakai (RIKEN) and the staff at the BL26B2 beamline (Proposal No. 20190047) of SPring-8 (Harima, Japan) and the X06DA beamline (Proposal No. 20171001) of the Swiss Light Source, Paul Scherrer Institut (Villigen, Switzerland) for their help in X-ray diffraction data collection. We acknowledge support from the National Institutes for Health (grant nos. 5R01GM123130 to T.I., and 5R01GM123247 and 1R35 GM134864 to N.V.D.), the Passan Foundation to N.V.D., the DoD DARPA (grant no. HR0011-16-C-0139 to T.I.), and the PRESTO program of the Japan Science and Technology Agency to T.U. (grant no. JPMJPR12A3) and T.I. (grant no. JPMJPR12A5) and a Grant-in-Aid for Scientific Research (B) to T.U. (grant no. 16H05089) from the Japan Society for the Promotion of Science.

Author information

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Authors

Contributions

H.D.W. and H.N. conceived the study with input from T.I. H.D.W. carried out cell experiments and conducted image analysis with help from A.K.A. M.K. purified and crystalized split proteins, and determined protein structure by X-ray crystallography. O.D. conducted rational split site analysis. T.I., H.N., T.U. and N.V.D. supervised the project. H.D.W. wrote the manuscript in consultation with T.I. and with input from M.K., T.U., O.D. and N.V.D.

Corresponding authors

Correspondence to Takashi Umehara or Takanari Inoue.

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Competing interests

There is an ongoing disclosure associated with the CIT tools.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Negative controls for cytosolic FKBP recruitment to ER-PM MCS.

a, Assessing contributions of each CIT component in CFP-FKBP recruitment to ER-PM MCS; left, middle and center panels correspond to constructs lacking ER, PM, and cytosolic CIT components. b, Quantifying trimerization between the 3 signals pre- and post- 100 nM rapamycin. Check marks specify each combination of two wavelengths used in calculating pairwise Fisher’s transformation of Pearson’s correlation coefficients. Data are presented as mean values +/− SD. Two-tailed Student’s t-test assuming equal variance was used to compare correlations pre- and post-rapamycin. Fom left to right: n = 38, 27, and 32 cells; 3 independent experiments each. Scalebar, 10 μm. (***/****; p < 0.001/0.0001). Source data

Extended Data Fig. 2 sFKBP-based CIT recruitment of cytosolic FRB to ER-PM MCS.

(a, b) Recruitment of mCh-FRB to ER-PM junctions by ER and PM targeted sFKBP1N and sFKBP1C, pre- and 1 h post- 100 nM rapamycin addition. FRB-mCh recruitment can be (a) undetectable or (b) prominent. (c) Quantifying trimerization between the 3 signals pre- and post-rapamycin. Check marks specify each combination of two wavelengths used in calculating pairwise Fisher’s transformation of Pearson’s correlation coefficients. Data are presented as mean values +/- SD. Two-tailed Student’s t-test assuming equal variance was used to compare correlations pre- and post-rapamycin. From left to right: n = 24 cells; 3 independent experiments each. Scalebar, 10 μm. (****; p < 0.0001). Source data

Extended Data Fig. 3 Negative controls for cytosolic FKBP recruitment to ER-mitochondria MCS.

a, Assessing contributions of each CIT component in CFP-FKBP recruitment to ER-mitochondria MCS; left, middle and center panels correspond to constructs lacking ER, mitochondria, and cytosolic CIT components. b, Quantifying trimerization between the 3 signals pre- and post- 100 nM rapamycin. Check marks specify each combination of two wavelengths used in calculating pairwise Fisher’s transformation of Pearson’s correlation coefficients. Data are presented as mean values +/− SD. Two-tailed Student’s t-test assuming equal variance was used to compare correlations pre- and 12 mins post-rapamycin. From left to right: n = 32, 26, and 24 cells; 3 independent experiments each. Scalebar, 10 μm. (****; p < 0.0001). Source data

Extended Data Fig. 4 Negative controls for CIT-induced ER-mitochondria-PM tri-organellar membrane contact sites (MCS).

a, Assessing contributions of each CIT component in tri-organellar MCS formation; left, middle and center panels correspond to constructs lacking ER, mitochondria, and PM CIT components. b, Quantifying trimerization between the 3 signals pre- and post- 100 nM rapamycin. Check marks specify each combination of two wavelengths used in calculating pairwise Fisher’s transformation of Pearson’s correlation coefficients. Data are presented as mean values +/- SD. Two-tailed Student’s t-test assuming equal variance was used to compare correlations pre- and 15 mins post-rapamycin. From left to right: n = 24, 32 and 28 cells; 3 independent experiments each. Scalebar, 10 μm. (****; p < 0.0001). Source data

Extended Data Fig. 5 PIP2 biosensor intensity at ER-PM junction sites over time.

Intensities of mRuby–PH-PLCδ PIP2 biosensor of user defined regions of interest inside and outside ER-PM MCS at 1, 2, 3, 4 and 10 min after 100 nM rapamycin. Significance analyzed with two-tailed paired Students t-tests assuming equal variance. From left to right: n = 28, 25 and 28 cells; 4 independent experiments each. (*/***/****; p < 0.05/0.001/0.0001). Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–7

Reporting Summary

Supplementary Video 1

Timelapse epifluorescence images of Cos-7 cells showing recruitment of cytosolic FKBP (magenta) to ER–PM junction sites with CIT on rapamycin addition. ER in cyan, PM in yellow. Constructs expressed: sFRB1N–CFP–Cb5/Tom20–YFP–sFRB1C/mCh–FKBP. Time in mm:ss; scale bar, 10 μm.

Supplementary Video 2

Timelapse epifluorescence images of Cos-7 cells showing recruitment of cytosolic FRB (magenta) to ER–PM junction sites with CIT on rapamycin addition. ER in cyan, PM in yellow. Constructs expressed: CFP–FKBP1C-Cb5/Lyn–YFP–sFKBP1N/FRB–mCh. Time in mm:ss; scale bar, 10 μm.

Supplementary Video 3

Timelapse epifluorescence images of Cos-7 cells showing recruitment of cytosolic FKBP (magenta) to ER–mitochondria junction sites with CIT on rapamycin addition. ER in cyan, mitochondria in yellow. Constructs expressed: mCh–sFRB1C–Cb5/Lyn–YFP–sFRB1N/CFP–FKBP. Time in mm:ss; scale bar, 10 μm.

Supplementary Video 4

Timelapse epifluorescence images of Cos-7 cells showing tri-organellar junction formation between ER, PM and mitochondria on rapamycin addition. Mitochondria in magenta, ER in yellow, PM in cyan. Constructs expressed: YFP–sFRB1N–Cb5/Tom20–mCh–FKBP/Lyn–CFP–sFRB1C. Time in mm:ss; scale bar, 10 μm.

Supplementary Video 5

Timelapse epifluorescence images of Cos-7 cells showing recruitment of INP54P (cyan) to ER–PM junctions resulting in reduced signal intensity of PH–PLCδ (gray). ER in magenta, PM in yellow. Constructs expressed: CFP–FKBP–INP54P(331)/iRFP–sFRB1C–Cb5/Lyn–Clover–sFRB1N/mRuby–PH–PLCδ. Time in mm:ss; scale bar, 10 μm.

Supplementary Video 6

Timelapse epifluorescence images of Cos-7 cells showing recruitment of INP54P D281A (cyan) to ER–PM junctions resulting in no change in signal intensity of PH–PLCδ (gray). ER in magenta, PM in yellow. Constructs expressed: CFP–FKBP–INP54P D281A/iRFP–sFRB1C–Cb5/Lyn–Clover–sFRB1N/mRuby–PH–PLCδ. Time in mm:ss; scale bar, 10 μm.

Supplementary Video 7

Timelapse epifluorescence images of Cos-7 cells showing recruitment of FKBP (cyan) to ER–PM junctions resulting in no change in signal intensity of PH–PLCδ (gray). ER in magenta, PM in yellow. Constructs expressed: CFP–FKBP/iRFP–sFRB1C–Cb5/Lyn–Clover–sFRB1N/mRuby–PH–PLCδ. Time in mm:ss; scale bar, 10 μm.

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Wu, H.D., Kikuchi, M., Dagliyan, O. et al. Rational design and implementation of a chemically inducible heterotrimerization system. Nat Methods 17, 928–936 (2020). https://doi.org/10.1038/s41592-020-0913-x

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